JP6541581B2 - Low concentration antibody preparation - Google Patents

Description

  The present invention relates to the field of formulations for therapeutic proteins. More specifically, the present invention relates to formulations for low concentrations of therapeutic proteins, and methods of manufacturing the formulations.

  Although many biopharmaceuticals have been formulated and provided that are ready for clinical administration without further manipulation, many products require varying degrees of handling by a nurse, pharmacist, or physician . During handling and administration, the physical and chemical stability of the protein should be maintained. The loss of stability can occur when the protein preparation is diluted to a low concentration in an intravenous solution (iv), resulting in a reduction of the excipient concentration and a change in the composition and properties of the original drug preparation There is. When delivering a biopharmaceutical product by the iv route, several factors must be considered, including protein characteristics, formulation composition, concentration of active product to be delivered, choice of diluent, interface, and time and rate of injection. It does not. Contact surfaces are of particular interest because proteins tend to adsorb at the interface due to their amphiphilic nature. Due to the widespread use of various plastic polymers in syringes and iv injection containers and lines, the risk of protein loss due to adsorption is particularly large at low concentrations (<0.5 mg / mL). Thus, especially during drug development for low dose products, protein loss due to adsorption to filters, containers, syringes and tubes must be investigated and addressed.

  The present invention provides formulations suitable for low concentration therapeutic proteins.

SUMMARY OF THE INVENTION In one aspect, the present invention provides a) a) and b): a therapeutic protein; and b) a surfactant; wherein the molar ratio of the surfactant to the therapeutic protein is at least 100. The present invention is directed to a formulation for said therapeutic protein, including

  In another aspect, the invention comprises the following a) and b): a) a therapeutic protein; and b) an antioxidant, wherein the molar ratio of said antioxidant to said therapeutic protein is at least 750 Certain formulations for the therapeutic protein are directed.

FIG. 1 shows the results of the average recovery (%) determined by Poros A-HPLC analysis. FIG. 2 shows the mean% recovery, as determined by Poros A-HPLC analysis. FIG. 3 shows non-reducing SDS-PAGE for mAb samples formulated with various antioxidants and stored for 2 weeks at 40 ° C. (differences observed in each sample containing antioxidants Various gel-derived lanes were combined to better illustrate). FIG. 4 shows mAb stability sample C (containing a combination of surfactant and two antioxidants), G (containing only surfactant), and H (containing only surfactant) stored for 6 months at 5 ° C. and 25 ° C. (Excipient-free control) shows the tendency of oxidation. FIG. 5 shows the Oteroxizumab heavy chain amino acid sequence (SEQ ID NO: 1). FIG. 6 shows the Oteroxizumab light chain amino acid sequence (SEQ ID NO: 2).

  It is to be understood that the present invention is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. Similarly, it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include plural referents unless the context clearly indicates otherwise. including. Thus, for example, reference to "a sugar" includes combinations of two or more sugars and the like.

  As used herein, “about” when referring to a measurable value, such as amount, time period, etc., ± 20% or ± 10%, eg ± 5%, ± 1 from the stated value Such variations are intended to be included, as variations of% and ± 0.1% are appropriate for carrying out the disclosed method.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention for testing, preferred materials and methods are described herein. It is described in the inside. In describing and claiming the present invention, the following terms will be used.

  In one embodiment, the present invention comprises the following a) and b): a) therapeutic protein; and b) surfactant; wherein the molar ratio of said surfactant to said therapeutic protein is at least 100: It is directed to a formulation for said therapeutic protein, including.

  In certain embodiments, the molar ratio of surfactant to therapeutic protein is selected from the group consisting of at least 150, at least 200, at least 250, at least 300, at least 400, and at least 500. In one embodiment, the molar ratio of surfactant to therapeutic protein is about 545.

  In certain embodiments, the surfactant is selected from the group consisting of polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer 188, and mixtures thereof Be done. In one embodiment, the formulation comprises about 0.01% w / v to about 0.5% w / v surfactant. In certain embodiments, about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, or about 0.5% w / v surfactant. In one embodiment, the formulation comprises about 0.1% w / v polysorbate 80.

  In certain embodiments, the present invention comprises the following: a) and b): a therapeutic protein; and b) an antioxidant, wherein the molar ratio of the antioxidant to the therapeutic protein is at least 750. It is directed to a formulation for said therapeutic protein, including.

  In certain embodiments, the molar ratio of antioxidant to therapeutic protein is at least 1000, at least 1250, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least It is selected from the group consisting of 5000, at least 5500, at least 6000, at least 6500, and at least 7000. In one embodiment, the molar ratio of antioxidant to therapeutic protein is about 7143. In certain embodiments, the antioxidant is selected from the group consisting of methionine, cysteine, glutathione, and monothioglycerol. In another embodiment, the antioxidant is a metal chelator. Metal chelating agents include ethylenediamine tetraacetate ("EDTA"), ethylene glycol tetraacetic acid ("EGTA"), thiamine tetrahydrofurfuryl disulfide ("TTFD"), and 2,3-dimercaptosuccinic acid ("DMSA") And the like, but is not limited thereto. In certain embodiments, the formulation comprises about 1 mM to about 50 mM of an antioxidant. In certain embodiments, the formulation is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 45 mM of an antioxidant including. In one embodiment, the formulation comprises about 10 mM methionine.

  In certain embodiments, the formulation further comprises a buffer, wherein the pH of the formulation is about 4.0 to about 8.0. In certain embodiments, the pH of the formulation is about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.2, about 7.5, or about 8.0. In one embodiment, the buffer is selected from the group consisting of histidine, acetate, succinate, and citrate. In certain embodiments, the formulation comprises about 1 mM to about 100 mM buffer. In certain embodiments, the formulation is about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM. Buffer. In one embodiment, the formulation comprises about 20 mM histidine at a pH of about 6.5.

  In certain embodiments, the therapeutic protein is an antigen binding protein. In one embodiment, the antigen binding protein is an antibody or a fragment thereof. In one embodiment, the antigen binding protein is an immunoglobulin single variable domain. In one embodiment, the antigen binding protein binds human CD3. In one embodiment, the antigen binding polypeptide is an anti-CD3 antibody. In one embodiment, the anti-CD3 antibody comprises a heavy chain comprising SEQ ID NO: 1 and a light chain comprising SEQ ID NO: 2.

  In certain embodiments, the therapeutic protein is present at a concentration of about 0.01 mg / mL to about 1 mg / mL. In one embodiment, the therapeutic protein is present at a concentration of about 0.1 mg / mL to about 0.5 mg / mL. In one embodiment, the therapeutic protein is present at a concentration of about 0.2 mg / mL.

  In one embodiment, the formulation is a reconstituted formulation. In one embodiment, the formulation is a liquid pharmaceutical formulation. In one embodiment, the formulation is suitable for parenteral administration.

  In certain embodiments, the present invention is a formulation for a therapeutic protein, comprising a) to d): a) a therapeutic protein; and b) about 0.01% w / v to about 0.5% w / v Surfactant, wherein the molar ratio of said surfactant to said therapeutic protein is at least 100; c) from about 1 mM to about 50 mM of an antioxidant, wherein said molar ratio of said antioxidant to a therapeutic protein And d) comprising about 1 mM to about 100 mM of a buffer, and wherein the pH of the formulation is about 4.0 to about 8.0.

  In one embodiment, the formulation further comprises about 0.01 mM to about 1.0 mM EDTA. In certain embodiments, the formulation is about 0.05 mM, about 0.1 mM, about 0.15 mM, about 0.2 mM, about 0.25 mM, about 0.3 mM, about 0.35 mM, about 0.4 mM, about 0.45 mM, about 0.5 mM, About 0.55 mM, about 0.6 mM, 0.65 mM, about 0.7 mM, about 0.75 mM, about 0.8 mM, about 0.85 mM, about 0.9 mM, about 0.95 mM, or about 1.0 mM EDTA. In one embodiment, the formulation comprises about 0.05 mM EDTA.

  "Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The polypeptide may be of natural (tissue-derived) origin, recombinant or naturally expressed from a prokaryotic or eukaryotic cell preparation, or chemically produced by synthetic methods. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Non-naturally occurring residues are well described in the scientific and patent literature; few exemplary non-natural components and guidelines useful as mimetics of natural amino acid residues are described below. Mimetics of aromatic amino acids are, for example, D- or L-nafilalanine (naphylalanine); D- or L-phenylglycine; D- or L-2 thienailalalanine (thieneylalanine); D- or L-1,- 2,3- or 4-pyrenailalalanine; D- or L-3 thienailalalanine; D- or L- (2-pyridinyl) -alanine; D- or L- (3-pyridinyl) D- or L- (2-pyrazinyl) -alanine; D- or L- (4-isopropyl) -phenylglycine; D- (trifluoromethyl) -phenylglycine; D- (trifluoromethyl) -phenylalanine D-p-fluoro-phenylalanine; D- or L-p-biphenyl phenylalanine; K- or L-p-methoxy-biphenyl phenylalanine; D- or L-2 indole (alkyl) alanine; and D- or L-alkyl Substitute by alanine (alkylalanine) Can be prepared by reaction, where alkyl is substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, secondary-isotyl, iso-pentyl or non- It may be an acidic amino acid. As an aromatic ring of a nonnatural amino acid, for example, thiazoyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl and pyridyl aromatic ring can be mentioned.

  As used herein, "peptide" includes peptides that are conservative variations of the peptides specifically exemplified herein. As used herein, "conservative mutation" refers to the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative mutations include, but are not limited to, one hydrophobic residue such as, but not limited to isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine, or methionine. Substitution of hydrophobic residues or substitution of one polar residue with another such as substitution of arginine with lysine, substitution of glutamic acid with aspartic acid, or substitution of glutamine with asparagine, etc. Can be mentioned. Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine and threonine. "Conservative mutation" also includes using a substituted amino acid in place of a non-substituted parent amino acid, as long as the antibody raised against the substituted polypeptide also produces an immune response with the non-substituted polypeptide. Such conservative substitutions are within the definition of the class of peptides of the invention. As used herein, "cationic" means any peptide having a net positive charge at pH 7.4. The biological activity of the peptide is known to the person skilled in the art and can be measured by the standard methods described herein.

  "Recombinant" when used in connection with a protein refers to a protein that has been modified by the introduction of heterologous nucleic acids or proteins or by changes in natural nucleic acids or proteins.

  As used herein, a "therapeutic protein" can be administered to a mammal to elicit, for example, a biological or medical response in a tissue, system, animal, or human that a researcher or physician seeks Refers to any protein and / or polypeptide. Therapeutic proteins can cause more than one biological or medical response. Furthermore, the term "therapeutically effective amount" refers to, but is not limited to, the cure, prevention, or amelioration of the disease, disorder, or side effects, as compared to a corresponding subject who has not received such an amount. Or any amount that results in a reduction in the rate of progression of the disorder. Also within the scope of this term is an amount that is effective to improve normal physiological function, as well as an amount that is effective to trigger a patient's physiological function that enhances or supplements the therapeutic effect of the second pharmaceutical agent. Including.

All "amino acid" residues specified herein are in the natural L-configuration. In accordance with standard polypeptide nomenclature, abbreviations for amino acid residues are shown below.

  It should be noted that all amino acid residue sequences are represented herein by a formula whose left to right orientation is the conventional amino terminus to carboxy terminus orientation.

  In another embodiment, the polypeptide is an antigen binding polypeptide. In one embodiment, the antigen binding polypeptide comprises a soluble receptor, antibody, antibody fragment, immunoglobulin single variable domain, Fab, F (ab ') 2, Fv, disulfide linked Fv, scFv, closed conformation multispecific It is selected from the group consisting of a sex antibody, a disulfide linked scFv, or a bispecific antibody.

  As used herein, the term "antigen binding polypeptide" refers to antibodies, antibody fragments, and other protein constructs that have the ability to bind antigen.

  The terms "Fv, Fc, Fd, Fab or F (ab) 2" are used in their standard sense (see, for example, Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory (1988). ).

  A "chimeric antibody" refers to a type of genetically engineered antibody that comprises naturally occurring variable regions (light and heavy chains) derived from a donor antibody in combination with light and heavy chain constant regions derived from an acceptor antibody.

  A "humanized antibody" refers to a type of genetically engineered antibody that has CDRs from non-human donor immunoglobulins and the remaining immunoglobulin-derived portion of the molecule is derived from one (or more) human immunoglobulins. In addition, framework support residues may be modified to preserve binding affinity (eg, Queen et al., Proc. Natl. Acad Sci USA, 86: 1029-10032 ( 1989), Hodgson et al., Bio / Technology, 9: 421 (1991)). Suitable human acceptor antibodies can be selected by homology to the nucleotide and amino acid sequences of the donor antibody from conventional databases, such as the KABAT.RTM. Database, the Los Alamos database, and the Swiss Protein database. Human antibodies characterized by homology (based on amino acids) to the framework region of the donor antibody are suitable to provide a heavy chain constant region and / or a heavy chain variable framework region for insertion of donor CDRs. obtain. Suitable acceptor antibodies that can provide light chain constant or variable framework regions can be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains need not be from the same acceptor antibody. The prior art describes several methods for such humanized antibody production, see, for example, EP 0 239 400 A and EP 0 495 51 A.

  The term "donor antibody" provides the amino acid sequence of its variable region, CDRs, or other functional fragments, or analogs thereof, to a first immunoglobulin partner, thereby providing a modified immunoglobulin coding region. And as a result, it refers to an antibody (monoclonal and / or recombinant) that provides for expression of a modified antibody having antigen specificity and neutralizing activity that is characteristic of the donor antibody.

  The term "acceptor antibody" is heterologous to the donor antibody and comprises all of the amino acid sequences encoding its heavy and / or light chain framework regions and / or its heavy and / or light chain constant regions (or Refers to an antibody (monoclonal and / or recombinant) that provides any portion, but in some embodiments, to a first immunoglobulin partner. In certain embodiments, the human antibody is an acceptor antibody.

  "CDR" is defined as the complementarity determining region amino acid sequence of an antibody, which is the hypervariable region of an immunoglobulin heavy and light chain (e.g. Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., See US Department of Health and Human Services, National Institutes of Health (1987)). There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable part of the immunoglobulin. Thus, as used herein, “CDR” refers to all three heavy chain CDRs, or all three light chain CDRs (or, where appropriate, both all heavy chain CDRs and all light chain CDRs) Means The structure of the antibody and protein folding can mean that other residues are considered to be part of the antigen binding region, and will be so understood by those skilled in the art. See, for example, Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions, Nature 342, p 877-883.

  As used herein, the term "domain" refers to a folded protein structure having a tertiary structure independent of the rest of the protein. In general, domains are responsible for the separate functional properties of a protein, and can often be added, removed or transferred to other proteins without loss of the protein and / or the remaining function of that domain . An "antibody single variable domain" is a folded polypeptide domain that comprises sequences characteristic of antibody variable domains. Thus, it may be a complete antibody variable domain, and a modified variable domain, eg, one or more of the loops replaced by sequences not characteristic of the antibody variable domain, or truncated, or N-terminal or C-terminal An antibody variable domain comprising an extension, and a folded fragment of the variable domain which maintains the binding activity and specificity of at least the full length domain.

The phrase "immunoglobulin single variable domain" means an antibody variable domain (V _H , V _HH , V _L ) that specifically binds to an antigen or epitope independently of the different V regions or domains. The immunoglobulin single variable domain may be present as a format (e.g. homodimer or heteromultimer) with another different variable region or variable domain, wherein the other region or domain is an antigen according to a single immunoglobulin variable domain It is not necessary for binding (ie, the immunoglobulin single variable domain binds the antigen independently of the additional variable domain). A "domain antibody" or "dAb" as the term is used herein is the same as an "immunoglobulin single variable domain" capable of binding an antigen. The immunoglobulin single variable domain may be a human antibody variable domain, but is disclosed (for example, as disclosed in WO 00/29004) rodents, common sharks, and camelid (Chamelid) V _HH dAbs (nanobodies) Also includes single antibody variable domains from other species, etc. Camelid V _HH are originally immunoglobulin single variable domain polypeptide is a species derived from a light chain without camels which produce heavy chain antibodies, llama, alpaca, dromedary, and guanaco. Such V _HH domains can be humanized according to standard techniques available in the art and such domains are still considered as "domain antibodies" in the present invention. As used herein, V _H includes camelid V _HH domains. NARV is another type of immunoglobulin single variable domain that has been identified in cartilaginous fish, including common sharks. These domains are also known as Novel Antigen Antigen Receptor variable regions (generally abbreviated as V (NAR) or NARV). For further details, see Mol. Immunol. 44, 656-665 (2006) and U.S. Patent Application No. 20050043519A.

  The term "epitope binding domain" refers to a domain that specifically binds to an antigen or epitope independently of different V regions or domains, which is a domain antibody (dAb) such as human, camelid or shark immunity It may be a globulin single variable domain.

As used herein, the term "antigen binding site" means a site on a protein capable of specifically binding to an antigen, which may be a single domain, such as an epitope binding domain, or This may be a paired V _H / V _L domain that can be found on standard antibodies. In some aspects of the invention, a single chain Fv (ScFv) domain can provide an antigen binding site.

  The terms "mAb dAb" and "dAb mAb" are used herein to refer to the antigen binding protein of the invention. These two terms may be used interchangeably and are intended to have the same meaning as used herein.

In another embodiment, the formulation further comprises an additional excipient. As the "excipient", but not limited to, stabilizers, such as human serum albumin (hsa), bovine serum albumin (bsa), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin , Ovalbumin, RNase A; buffer, for example, citric acid, HEPES, histidine, potassium acetate, potassium citrate, potassium phosphate (KH ₂ PO ₄ ), sodium acetate, sodium hydrogencarbonate, sodium bicarbonate, sodium citrate, phosphoric acid Sodium (NAH ₂ PO ₄ ), Tris base, and Tris-HCl; amino acids / metabolites such as glycine, alanine (α-alanine, β-alanine), arginine, betaine, leucine, lysine, glutamic acid, aspartic acid, histidine , Proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opine (alanopine, octopine, stronbi ), And trimethylamine N-oxide (TMAO); surfactants such as polysorbate 20 and 80, and poloxamer 407: lipid molecules such as phosphatidylcholine, ethanolamine, and acetyltriptophanate: polymers such as polyethylene glycol (PEG) And polyvinylpyrrolidone (PVP) 10, 24, 40; low molecular weight excipients such as arabinose, cellobiose, ethylene glycol, fructose, fucose, galactose, glycerol / glycerol, glucose, inositol, lactose, maltose, maltotri Aus, mannose, melibiose, 2-methyl-2,4-pentanediol, octulose, propylene glycol, raffinose, ribose, sucrose, trehalose, xylitol, and xylose; High molecular weight excipients such as cellulose, β-cyclodextrin, dextran (10 kd), dextran (40 kd), dextran (70 kd), ficoll, gelatin, hydroxypropyl methyl-cellulose, hydroxyethyl starch, Maltodextrin, Methocel, peg (6 kd), polydextrose, polyvinyl pyrrolidone (PVP) k15 (10 kd), PVP (40 kd), PVP k30 (40 kd), PVP k90 (1000 kd), Sephadex G 200, And starches; antioxidants such as ascorbic acid, cysteine HCl, thioglycerol, thioglycollic acid, thiosorbitol, and glutathione; reducing agents such as cysteine HCl, dithiothreitol and other thiols or thiophenes; chelating agents, For example, EDTA, EGTA, glutamic acid and aspartic acid; inorganic salts / metals such as Ca ²⁺ , Ni ²⁺ , Mg ²⁺ , Mn ²⁺ , Na ₂ SO ₄ , (NH ₄ ) ₂ SO ₄ , Na ₂ HPO ₄ / NaH ₂ PO ₄ , K ₂ HPO ₄ / KH ₂ PO ₄ , MgSO ₄ , and NaF; Salts, such as sodium acetate, polyethylene Na, sodium caprate (sodium octanoate), propionate, lactate, succinate, and citrate; organic solvents such as, for example, acetonitrile, dimethyl sulfoxide (dmso), and ethanol Can be mentioned.

  In one embodiment, the formulation is formulated to a pH of about 4.0 to about 8.0. In one embodiment, the formulation is formulated to a pH of about 6.5. In one embodiment, the formulation comprises about 10 mM to about 50 mM histidine. In one embodiment, the formulation comprises about 20 mM histidine.

  The agents used to stabilize the therapeutic protein can be added at any stage of the formulation process. For example, it can be added before, after, or at the same time as the aforementioned buffer, therapeutic protein, or together with an optional excipient.

  The formulations of the invention may be administered by any suitable route of administration, including systemic administration. Systemic administration includes oral, parenteral, transdermal, rectal, and by inhalation. Parenteral administration refers to routes of administration other than enteral, transdermal, or by inhalation, and typically refers to injection or infusion. Parenteral administration includes intravenous, intramuscular and subcutaneous injection or infusion. Inhalation refers to administration to the patient's lungs, whether inhaled through the mouth or through the nasal cavity.

The invention is also directed to stable formulations made by any of the methods of the invention.
(Example)

Example 1-Oteroxizumab is a Low Concentration-Low Dose mAb Oterixizumab is a humanized mAb (IgG1) to human CD3 that has no glycosylation site in its Fc domain. To facilitate accurate preparation and administration of clinical doses (0.1-0.5 mg per day), this drug is made at a concentration of 0.2 mg / mL using different vial unit indications corresponding to different daily doses did. Administration of the dose required dilution of the mAb in an iv bag containing isotonic 0.9% saline (saline) and delivery of the entire bag contents by a standard infusion pump. This dosing method resulted in protein concentrations in the iv bag as low as 2 μg / mL. As a direct consequence, the risk of significant protein loss was very high. For these reasons, the proposed dilutions and evaluations of the administration feasibility of existing products by performing short-term stability and compatibility studies with commercially relevant iv administration sets, as well as the proposed dilution and An alternative formulation was developed that would support the intent of dosing design.

Example 2-Evaluation of unacceptable mAb loss during in-use stability studies To evaluate the feasibility of administering the above 0.2 mg / mL formulation consisting of histidine buffer only, using a commercial iv dosing set The stability test was performed. An amount of drug equivalent to the target dose (0.1 mg) was added to the iv saline bag and the stability of this diluted product was assessed. When a relatively small saline volume of 25 or 50 mL was chosen to minimize potential protein loss due to adsorption, the active concentration in the iv bag was 4 and 2 μg / mL, respectively. In addition, an iv bag composed of two different types of materials (polyolefin PO and polyvinyl chloride PVC) was evaluated. The bag was placed on a laboratory bench and exposed to ambient temperature and lighting conditions. Three sets of bags were prepared for each test configuration: bag 1 serves as a test of initial conditions, bag 2 represents hold, and bag 3 comprises an iv line (with in-line filter) B. It was used to simulate the clinical preparation and infusion of protein solutions through the Braun (Horizon Pump Filtered iv set). The bag was prepared and tested in duplicate. Samples were collected and tested immediately after iv bag preparation (retention in iv bag for 0 hours) and after 2 hours in iv bag. Additional samples were collected after 2 hours infusion through the iv line. Polysorbate 80 (PS80) was added to all holding samples to a final concentration of 0.001% to prevent loss due to adsorption in the collection tube. Average recovery (%) The results were determined by Poros A-HPLC analysis. The recovery was calculated based on the concentration of target in the bag and the reported value was the average of two different preparations.

  The average recovery (%) results determined by Poros A-HPLC analysis are plotted in FIG. Recovery in all PO bags (range 23% to 88%) tended to be lower than recovery in PVC bags (range 41% to 98%). Overall, the loss in all test systems after 2 hours of room temperature storage was well over 10%. Furthermore, when the product was injected through an iv line and filter, more than 50% of the protein was lost regardless of the bag material.

Example 3-Optimization of surfactant concentration in iv bag to improve stability to loss due to adsorption during dosing due to unacceptable protein recovery upon administration of mAb formulated with histidine buffer alone Then, a second study was performed to determine if incorporation of surfactant polysorbate 80 (PS 80) into the formulation could allow for the delivery of 90% or more of the dose. Thus, for preparation and injection of 0.1 mg of protein, PO bags containing 50 mL of saline were tested using in combination with an iv line equipped with an in-line filter (2 μg / mL mAb). Initial results show lower recovery in PO bags as compared to recovery in PVC bags, which may be a more difficult obstacle for PO bags to demonstrate acceptable protein recovery As it shows, I examined only this PO bag.

  A similar experimental design was used in this second study as follows: mAbs at a concentration of 0.2 mg / mL and containing various concentrations of PS80 (0.02, 0.05, 0.1, and 0.3% w / v) Stock solutions were freshly prepared and added to iv infusion bags (PO only) as described above. The final mAb concentration in this iv saline bag was 2 μg / mL, and the final surfactant concentrations were 0.0002, 0.0005, 0.001, and 0.003%. Additional PS80 (to a final concentration of at least 0.001%) was added to all samples prior to analysis. The iv line tested was a Baxter Continu-Flo Solution Set equipped with a filter. The mAb concentration was measured initially and after infusion for two hours with a hold in an iv bag or through a selected iv line. Recovery results were determined by Poros A-HPLC analysis. The recovery was calculated based on the concentration of target in the bag and the reported value was the average of two different preparations.

  The average% recovery determined by Poros A-HPLC analysis is plotted in FIG. Depending on the test conditions, the measured value is 66% to 105%. Approximately 95% protein recovery at all tested PS80 concentrations was observed in the 2 hour pending iv bag. Thus, a PS80 concentration as low as 0.0002% in the bag (equivalent to 0.02% in the drug formulation) was sufficient to suppress the loss of adsorption mediatedness of the mAb. However, as shown in FIG. 2, additional significant losses on iv lines and filters are observed. The overall protein loss dependence on surfactant concentration was considered to be non-linear, with nearly 90% recovery achieved at PS80 concentrations of 0.001% (or 0.1% in product) in 50 mL iv bags.

Example 4-Stability of PS80-Containing Products: Assessment of Oxidation Risk Protein stability under accelerated conditions is traditionally not only to give a prediction of shelf life, but also to select the optimal formulation for long-term storage Is also used in the development of biopharmaceuticals. Here, in order to evaluate the stability of the PS80-containing preparation, specifically, to evaluate the risk that PS80 may adversely affect the stability of mAb by oxidative degradation during long-term storage, 2) Accelerated stability test was conducted.

  The desired formulations were prepared by diluting the mAb drug substance with various PS80 stock solutions prepared in histidine buffer. The final mAb concentration in each vial was 0.2 mg / mL. Each test condition was equally assigned to multiple glass vials, and the vials were further stored at 40 ° C. for 2 weeks. Analytical tests included analysis by size exclusion chromatography ("SEC"), capillary isoelectric focusing ("cIEF"), and mass spectrometry ("MS").

  Oxidation of methionine residues results in the formation of methionine sulfoxide and a mass shift of 16 Da which can be detected using MS. The relative amounts of oxidized and nonoxidized peptides were compared by using MS. The results of MS are described in Table 1. For the mAb control sample (in excipient free buffer), the measured% oxidation at the methionine residue tested was as high as 11%. When surfactant was present in the formulation, significantly elevated levels of mAb oxidation (up to 30%) at the two most exposed methionine residues were observed. This increase in oxidation is a precursor to long term stability loss as oxidation can further cause aggregation and other secondary degradation processes.

Example 5-Enhanced Stability of PS80-Containing Products in the Presence of Selected Antioxidants Screening of Excipients That Will Improve Chemical Stability of the mAb in the Proposed 0.1% PS80-Containing Formulation In order to do that, we did additional research. Five different antioxidants, including multiple free radical scavengers and one metal chelator, were tested.

  Formulations of interest were prepared in histidine buffer, various excipient stock solutions as follows: PS80 (polysorbate 80); Met (methionine); Cys (cysteine); Glut (glutathione); MTG (monothioglycerol) ); Prepared by diluting mAb drug substance with EDTA (disodium edetate). The final antioxidant concentrations in this product vial were as follows: 5 mM Met, 5 mM Cys, 5 mM Glut, 5 mM MTG, and 1 mM EDTA. The final mAb concentration in each vial was 0.2 mg / mL. Each test condition was equally assigned to a plurality of glass vials, and the vials were further stored at 40 ° C. for 2 weeks. Analytical tests included analysis by cIEF and MS.

  As seen in Table 2, all antioxidants were able to attenuate the negative stability impact of the surfactant. The data show the lowest degree of oxidation in formulations containing methionine. Although EDTA appeared to be less effective than Met in controlling oxidation, it still significantly reduced the degree of PS80-dependent oxidation by nearly 4-fold.

Table 2 also shows the results of charge analysis by capillary isoelectric focusing (cIEF) analysis. Maximum% values were observed for the control formulations that did not contain antioxidants, and also for PS80-containing formulations that additionally contained Met or EDTA. The formulation with the least stability, as assessed using cIEF, contained Cys, Glut, or MTG.
Table 2
Stability of 0.2 mg / mL mAb formulated with 0.1% PS80 and various antioxidants: Results of MS and cIEF analysis after storage for 2 weeks at 40 ° C. in a vial

  SDS-PAGE analysis was also performed to help explain the discrepancy between MS and cIEF results. The SDS-PAGE results for formulations containing Cys, Glut, or MTG show a significant shift of bands in non-reduced gels that is indicative of chemical degradation by disulfide shuffling for samples stored for 2 weeks at 40 ° C. Shown (Figure 3). Chemical instability of the mAb in the presence of Cys, Glut, or MTG detected by SDS-PAGE correlated well with the increase of the acidic peak by cIEF (Table 2). Thus, Met and EDTA were the only two tested antioxidants that did not show disruption of the disulfide bond pattern in the product, and were also confirmed to have optimal antioxidant activity. The overall product stability of formulations containing antioxidants can be ranked as follows: Met> EDTA >> Cys, Glut, MTG.

Example 6-Confirmation of long-term stability and functional antioxidant levels of the reformulated product
A development stability study was conducted to evaluate the long-term and accelerated stability of 0.2 mg / mL mAb formulations containing PS80, Met, and EDTA. A wide range of conditions were tested, including a series of 6 Met concentrations ranging from 0-40 mM. EDTA levels were fixed at 0.05 mM based on previous experience with mAbs. A total of eight preparations were placed in stable condition. A summary of the tested formulations is shown in Table 3. Formulations AF incorporated 0, 5, 10, 15, 20, and 40 mM Met with fixed levels of EDTA (0.05 mM) and PS80 (0.1%). Formulations G and H served as controls for PS80 (alone) and buffer only (no excipient), respectively. Each formulation was aliquoted into multiple glass vials, and the vials were stored inverted under refrigerated (2-8 ° C., primary) and 25 ° C. (accelerated) conditions. The mAb concentration in each vial was 0.2 mg / mL. Analytical pulls were planned for 1, 2, 3 and 6 months and the results were compared with the initial.
Table 3
Experimental configuration of formulation developmental stability test. Detailed formulation composition related to each prescription code

  Oxidation levels for stability were measured using MS. Table 4 lists the oxidation levels of methionine residues determined by MS quantification following tryptic digestion of proteins and HPLC separation of the resulting peptides. Representative oxidation trends of samples C (PS80, Met, and EDTA), G (PS80 alone), and H (without excipient) stored at 2-8 ° C. and 25 ° C. for 6 months are plotted in FIG. . Oxidation was initially observed at the most exposed (theoretically) most exposed methionine residue 254 (M254) in all samples. Interestingly, differences between samples were present at the time of initial analysis, and samples G and H showed ̃3% oxidation at the second exposed methionine residue 430 (M430). Samples A to F contained at least one antioxidant (Met) but not one of samples G and H; thus, these data represent storage stability Not only is it possible, but it may also reflect degradation during sample preparation and analysis. The protective effect of the combined addition of EDTA and Met was evident in samples B-F where both antioxidants were present. Specifically, oxidation at M254 remained low (≦ 4.1%) over a period of 6 months at 2-8 ° C. and 25 ° C., and little or no oxidation was observed at M430. These data demonstrate the effectiveness of combining antioxidants.

  MS analysis of EDTA-free, Met-free sample A helped to identify the various oxidation mechanisms. Although the oxidation level in sample A remained slightly lower than that in control sample H (buffer alone) and much lower than the oxidation level in control sample G (PS80), they added to EDTA Were higher than the oxidation levels measured in samples B-F containing Met. The previous three hypotheses from the corresponding MS results: 1. Oxidation is a degradation pathway even in the absence of PS80, 2. Oxidation occurs during cold storage, and accelerates significantly at elevated storage temperatures, 3. The addition of PS80 probably accelerated the oxidative degradation pathway via both peroxide- and metal-dependent pathways, so that control sample G (PS80) and H (buffer, no excipient) Comparison with was even more critical. Data collected on samples stored for 6 months at 25 ° C. indicated that oxidation reached the highest level (100%) in the PS80 control formulation (G), as expected. In addition, the oxidation level detected in formulation (H) was significantly higher than the oxidation level in samples (A-F) containing the combination of antioxidants. These data show that the combination of the two antioxidants Met (free radical scavenger) and EDTA (metal chelating agent) essentially prevents oxidation, and that its quenching mechanism is not temperature dependent. Is shown.

Table 4
Reported for various methionine residues in otelixizumab stability samples A to H after storage for 6 months at different storage temperatures, containing different levels of surfactant, antioxidants and metal chelating agents , Results of peptide mapping by MS analysis. The formulation composition associated with each prescription code is described in detail in Table 3.

Example 7-Composition of low concentration / low dose oteliximab formulation is a unique combination of two antioxidants at high surfactant level Table 5 below shows low concentration oteliximab formulation (0.2 mg / mL) It is a summary of the composition and excipient function of The level and functionality of the excipients are unique to this formulation.

Example 8-Comparison with Other Commercially Available mAb Formulations When compared to other commercial mAbs, the composition of Oterliximab described above exhibits high excipient / mAb ratio (s). See Table 6 below.

  For surfactant-containing Oterliximab formulations (0.2 mg / mL mAb and 0.1% PS80), the PS80 / mAb ratio is about 545. By comparison, eculizumab (Solilith) formulated at a concentration of 10 mg / mL mAb and 0.22% PS80 has a corresponding PS80 / mAb ratio of about 25. For another mAb, rituximab, the PS80 concentration is 0.07%; therefore, for a 10 mg / mL product, a PS80 / mAb ratio of approximately 8 can be calculated. In any of these cases, the surfactant is in molar excess; however, the excess is much smaller than the proposed ratio for oteliximab (545). At such a large molar excess of surfactant, the purity and chemical stability of the surfactant itself must be taken into consideration as it correlates with the stability of the mAb molecule.

For the antioxidant-containing oteliximab formulations tested (0.2 mg / mL mAb and 5 mM Met), the Met / mAb ratio was approximately 3,750. In the literature, an optimal ratio of 5 has been reported for the HER2 mAb. There are also examples of other parenteral biopharmaceutical formulations where the Met / mAb ratio is about 100 (follitropins alpha and beta). Although our ratio far exceeds that found in some commercial products, higher Met concentrations in our formulations may be directly correlated with higher PS80 concentrations. And may also be correlated with high PS80 / mAb ratios .
[1] A therapeutic protein of the following including a) and b): a) a therapeutic protein; and b) a surfactant, wherein the molar ratio of the surfactant to the therapeutic protein is at least 100. Formulation for.
[2] The preparation according to 1, wherein the molar ratio of surfactant to therapeutic protein is selected from the group consisting of at least 150, at least 200, at least 250, at least 300, at least 400, and at least 500.
[3] The preparation according to 1 or 2, wherein the molar ratio of the surfactant to the therapeutic protein is about 545.
[4] The surfactant is selected from the group consisting of polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer 88, and a mixture thereof, 1 to 4 The preparation according to any one of 3.
[5] The preparation according to any one of 1-4, wherein the preparation further comprises c) an antioxidant, wherein the molar ratio of the antioxidant to the therapeutic protein is at least 750.
[6] The preparation according to 5, wherein the molar ratio of the antioxidant to the therapeutic protein is selected from the group consisting of at least 5500, at least 6000, at least 6500, and at least 7000.
[7] The preparation according to 5 or 6, wherein the molar ratio of the antioxidant to the therapeutic protein is about 7143.
[8] The preparation according to any one of 5 to 7, wherein the antioxidant is selected from the group consisting of methionine, cysteine, glutathione and monothioglycerol.
[9] The preparation according to any one of 1-8, wherein the preparation further comprises d) a buffer, wherein the pH of the preparation is about 4.0 to about 8.0.
[10] The preparation according to 9, wherein the buffer is selected from the group consisting of histidine, acetate, citrate and succinate.
[11] The preparation according to any one of 1 to 10, wherein the therapeutic protein is an antigen binding protein.
[12] The preparation according to 11, wherein the antigen binding protein is an antibody or a fragment thereof.
[13] The preparation according to 11, wherein the antigen binding protein is an immunoglobulin single variable domain.
[14] The preparation according to 11, wherein the antigen binding protein binds to human CD3.
[15] The preparation according to 14, wherein the antigen-binding polypeptide is an anti-CD3 antibody.
[16] The preparation according to 15, wherein the anti-CD3 antibody comprises a heavy chain comprising SEQ ID NO: 1 and a light chain comprising SEQ ID NO: 2.
[17] The preparation according to any one of 1 to 16, wherein the therapeutic protein is present at a concentration of about 0.01 mg / mL to about 1 mg / mL.
[18] The preparation according to any one of 1 to 17, wherein the therapeutic protein is present at a concentration of about 0.1 mg / mL to about 0.5 mg / mL.
[19] The preparation according to any one of 1 to 18, wherein the therapeutic protein is present at a concentration of about 0.2 mg / mL.
[20] The preparation according to any one of 1 to 19, wherein the preparation is a reconstituted preparation.
[21] The preparation according to any one of 1 to 20, wherein the preparation is a liquid pharmaceutical preparation.
[22] The preparation according to any one of 1 to 21, wherein the preparation is suitable for parenteral administration.
[23] A formulation for a therapeutic protein, the following a) to d): a) therapeutic protein; and b) about 0.01% w / v to about 0.5% w / v of a surfactant, The molar ratio of the surfactant to the therapeutic protein is at least 100; c) an antioxidant of about 1 mM to about 50 mM, wherein the molar ratio of the antioxidant to the therapeutic protein is at least 750 And d) the above formulation, comprising about 1 mM to about 100 mM of buffer, wherein the pH of the formulation is about 4.0 to about 8.0.
[24] The formulation according to 23, wherein the therapeutic protein is an antibody, the surfactant is polysorbate 80, the antioxidant is methionine, and the buffer is histidine.
[25] The preparation according to 24, wherein the antibody is present at a concentration of about 0.2 mg / mL.
[26] The preparation according to any one of 1 to 25, wherein the preparation further comprises about 0.01 mM to about 1.0 mM EDTA.
[27] The preparation according to 26, wherein the preparation comprises about 0.01 mM to about 0.1 mM EDTA.
[28] The preparation according to 27, wherein the preparation comprises about 0.05 mM EDTA.

Claims (12)

A) and b) and c): a) a therapeutic protein which is an antigen binding protein, at a concentration of 0.01 mg / mL to 1 mg / mL; b) a surfactant; the molar ratio of the surfactant is at least 100, and c) an antioxidant, wherein the molar ratio of the antioxidant to said therapeutic protein is Ri least 750 der, the surfactant is polysorbate-20, formulations for polysorbate -40, polysorbate -60, polysorbate -65, polysorbate-80, polysorbate -85, the therapeutic protein comprising, Ru is selected from the group consisting of poloxamer 188, and mixtures thereof.   The formulation according to claim 1, wherein the molar ratio of surfactant to therapeutic protein is selected from the group consisting of at least 150, at least 200, at least 250, at least 300, at least 400, at least 500 and 545. The formulation according to claim 1 or 2 , wherein the molar ratio of antioxidant to therapeutic protein is selected from the group consisting of at least 5500, at least 6000, at least 6500, at least 7000 and 7143. The antioxidant is methionine, cysteine, is selected from the group consisting of glutathione, and monothioglycerol A formulation according to any one of claims 1-3. The formulation d) further comprises a buffer, wherein the pH of the formulation is 4.0 to 8.0, the formulation according to any one of claims 1-4. 6. The formulation according to claim 5 , wherein the buffer is selected from the group consisting of histidine, acetate, citrate and succinate. Wherein said antigen binding protein is an antibody or fragment thereof, is selected from the group consisting of an immunoglobulin single variable domain, and anti -CD3 antibody, said anti -CD3 antibody heavy chain and SEQ ID NO: 2 having the sequence of SEQ ID NO: 1 It comprises a light chain having the sequence a formulation according to any one of claims 1-6. The therapeutic protein is present at a concentration of 0.1 mg / mL~0.5 mg / mL or 0.2 mg / mL, the formulation according to any one of claims 1-7. Wherein the formulation is a formulation that is reconstituted and / or a liquid pharmaceutical formulation, and / or is suitable for parenteral administration, the formulation according to any one of claims 1-8. A formulation for a therapeutic protein, wherein the following a) to d): a therapeutic protein that is an antigen binding protein, at a concentration of 0.01 mg / mL to 1 mg / mL; and b) 0.01% w / v ~ 0.5% w / v surfactant, wherein the molar ratio of said surfactant to said therapeutic protein is at least 100; c) 1 mM to 50 mM antioxidant, here to therapeutic protein the molar ratio of the antioxidant is at least 750; and d) includes a buffer 1 mM~100 mM, wherein the pH of the formulation Ri der 4.0 to 8.0, wherein the surfactant is polysorbate-20 , polysorbate -40, polysorbate -60, polysorbate -65, polysorbate-80, polysorbate -85, Ru is selected from the group consisting of poloxamer 188, and mixtures thereof, said formulation. The preparation according to claim 10 , wherein the therapeutic protein is an antibody, the surfactant is polysorbate 80, the antioxidant is methionine, and the buffer is histidine. It said formulation further 0.01 mM~1.0 mM of EDTA, including 0.01 mM~0.1 mM of EDTA or 0.05 mM of EDTA,, formulation according to any one of claims 1 to 11.

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